Epigenetic Differences and Therapeutic Applications
Epigenetic changes alter gene expression, and are heritable and yet do not arise due to alterations of DNA sequence. Epigenetic differences allow two cells within the same organism, containing the same genetic complement of DNA, to express unique subsets of genes and to differentiate. Epigenetic differences are initiated and sustained by DNA methylation, RNA-associated silencing, or histone modifications. These components of epigenetic gene regulation can interact and stabilize each other, and as a result modulate chromatin structure. Disruption of one or more of these interacting systems can lead to inappropriate expression or silencing of genes, resulting in what are known as ‘epigenetic diseases’, which include developmental disorders such as fragile X syndrome, Angelman's syndrome, Prader-Willi syndrome, cancers such as leukemia, and immune regulation disorders such as ICF syndrome (Egger et al., Nature, 2004, 429, 457-463).
Methylation typically occurs at the cytosine of CpG dinucleotides within mammalian genomic DNA. “CpG islands” are long tracts of DNA with high GC content commonly found in promoter regions. In general, methylation of promoters is inhibitory and accounts for one type of epigenetic gene silencing, such as occurs in parentally imprinted genes. With the exception of the X-chromosome, CpG residues in promoter regions are typically unmethylated, and methylation occurs after DNA replication, resulting in a loss of gene expression (Gamis et al., Mol. Cancer, 2004, 3, 1-23).
Histone modifications occur post-transcriptionally and include the acetylation and methylation of conserved lysine residues on the amino-terminal tail domains of histones. Generally, the acetylation of histones marks active, transcriptionally competent regions, whereas under-acetylated histones are found in transcriptionally inactive euchromatic or heterochromatic regions. Histone methylation can be a marker for both active and inactive regions of chromatin. For example, methylation of lysine 9 on the N terminus of histone H3 (H3-K9) is a hallmark of silent DNA and is globally distributed throughout heterochromatic regions such as centromeres and telomeres. Conversely, methylation of lysine 4 of histone H3 (H3-K4) denotes activity and is found predominantly at promoters of active genes (Lachner and Jenuwein, Curr. Opin. Cell Biol., 2002, 14, 286-298).
RNA-associated silencing plays a role in post-transcriptional gene silencing and is accomplished by RNA in the form of antisense transcripts, non-coding RNAs, or RNA interference. RNA-associated silencing can also lead to mitotically heritable transcriptional silencing by the formation of heterochromatin.
Aberrant changes in the epigenetic control of gene expression have been increasingly recognized as factors contributing to the development of human disease, including hyperproliferative disorders, autoimmune disorders and developmental disorders. By way of example, aberrant, heritable gene silencing as a result of DNA hypermethylation has been linked to the genesis and progression of cancer. Inhibitors of DNA methylation, known as DNA demethylating agents, rapidly reactivate the expression of genes that have undergone epigenetic silencing, and are active only in S-phase cells. Likewise, the progression of cancer is often associated with epigenetic silencing associated with histone deacetylation, which is catalyzed by at least three classes of histone deacetylases (HDACs), thus HDAC inhibitors are used to induce differentiation, growth arrest and/or apoptosis in transformed cells in tumors (Egger et al., Nature, 2004, 429, 457-463).
While DNA demethylating drugs, such as 5-azacytidine, and HDAC inhibitors, such as phenylbutyric acid, are used to prevent or reverse hypermethylation, or acetylation, respective, such agents do not target individual enzymes or cell types. Thus, the application of demethylating agents or HDAC inhibitors to the treatment of disease lacks precision. The concerns regarding the clinical applications of these agents relate mainly to the nonspecific activation of genes and transposable elements in normal cells, and also to potential mutagenicity and carcinogenicity. Imprinted genes can be activated by 5-azacytidine, underscoring the need for careful application of this and related agents (Eversole-Cire, Mol. Cell. Biol., 1993, 13, 49284938).
Agents which work through RNA interference pathways are currently in research and development for possible therapeutic applications. There are concerns that these agents may induce specific or non-specific alterations in epigenetic control of gene expression. Thus, there is a need to ensure that any agents working through an RNA interference pathway that are developed for therapeutic applications do not cause undesired side effects through RNA associated silencing.
Heterochromatin also plays a role in the regulation of gene expression during development and cellular differentiation. Epigenetic control of gene expression may be a factor in the differentiation and dedifferentiation of pluripotent stem cells (such as embryonic stem cells) as well as in reprogramming the status of a cell during somatic cell nuclear transfer (i.e. cloning). Modulating the epigenetic control of gene expression in stem cells could be useful to keep stem cells in an undifferentiated state or in driving the stem cells towards a desired differentiated state.
As such, there remains a long-felt need for agents that regulate epigenetic processes such as DNA methylation, histone modifications and RNA-mediated silencing, without causing undesired alterations in the epigenetic control of gene expression. Similarly, there is a need for screening methods to identify agents that specifically regulate such epigenetic processes and allow for the development of therapeutic agents that modify aberrant epigenetic processes with precision. Oligomeric compounds targeting or mimicking specific small non-coding RNAs that participate in epigenetic processes are extremely attractive as therapeutic agents to selectively modulate epigenetic processes impacting human diseases such as cancers, developmental disorders, infections, and autoimmune disorders.
Mechanisms of Epigenetic Differences
Chromatin structure affects both gene transcription and cellular phenotype. The most condensed chromatin domains are known as heterochromatin, whereas the more extended chromatin domains are known as euchromatin. Euchromatic domains are generally transcriptionally active, accessible portions of the genome, whereas heterochromatic domains are generally inaccessible to DNA binding factors and are transcriptionally silent. Heterochromatin plays a role in chromosome structures, by stabilizing the repetitive DNA sequences at centromeres, telomeres and elsewhere in the genome and inhibiting recombination between homologous repeats. Furthermore, heterochromatin proteins associated with repeated DNA sequences surrounding centromeres are required for proper sister chromatid cohesion and chromosome segregation.
Faithful chromosome segregation and maintenance of genomic integrity are crucial cellular processes. For example, improper chromosome segregation during mitosis or meiosis results in aneuploidy, which is associated with tumorigenesis, spontaneous abortion, and congenital disorders such as trisomies (e.g., Down's syndrome). Telomere length is associated with cellular lifespan. Heterochromatin within centromeric and telomeric regions is believed to play both mechanical and regulatory roles in propagating and maintaining the integrity of the eukaryotic genome.
Heterochromatin-like structures are involved in the inactivation of developmental regulators such as the homeotic gene clusters in Drosophila and mammals, and the mating type genes in fungi. Moreover, dosage compensation in female mammals involves the heterochromatic inactivation of one of the two X chromosomes in somatic cells.
Another example of a heterochromatic region is an insulator. Insulators are DNA sequence elements that can, in some instances, act as barriers to protect a gene against the encroachment of adjacent inactive condensed chromatin. Insulators also can act as blocking elements to protect against the activating influence of neighboring cis-acting elements, and/or distal enhancers associated with other genes, for example, when the insulator is located between an enhancer and a promoter. Insulators thus are complex elements that can help to preserve the independent function of genes embedded in a genome in which they are surrounded by regulatory signals they must ignore.
Chromatin must be “remodeled” to allow transcription factors and RNA polymerase to interact with the DNA helix. Many of the trans-acting factors required for heterochromatin modulation are enzymes that directly modify histones. Histone modifying enzymes are known to regulate chromatin structure through acetylation, methylation, and/or phosphorylation of the histone proteins. In fission yeast and metazoans, several histone deacetylases (HDACs) are required for gene silencing. In fact, hypoacetylation works in conjunction with methylation to effect gene silencing. Methylation of a particular lysine in histone H3 (H3 K9) by the conserved histone methyltransferase Su(var)3-9 in Drosophila, SUV39H1 in human, and Clr4 in fission yeast creates a high affinity binding site within pericentric heterochromatin for the conserved heterochromatin protein 1 (HP1) in flies and humans, and the HP1-homolog in fission yeast, Swi6 (Grewel et al., Science, 2003, 301, 798-802; and Perrod et al., Cell. Mol. Life. Sci., 2003, 60, 2303-18). Furthermore, a complex containing both Suv39H1 and histone deacetylases is reported to be involved in heterochromatin silencing or transcriptional repression by the tumor suppressor retinoblastoma protein (Rb) (Vaute et al., Nucleic Acids Res., 2002, 30, 475-81). It has also been reported that cells entering mitosis with hyperacetylated histones displayed altered chromatin conformation associated with depletion of HP1 from the centromeric heterochromatin. Inhibition of histone deacetylation before mitosis produced defective chromosome condensation and impaired mitotic progression in living cells (Cimini et al., Mol. Biol. Cell, 2003, 14, 3821-3833). Thus, the methylation and acetylation states of histones may together direct centromeric heterochromatin formation.
Although chromatin compaction is necessary to contain huge lengths of DNA in the nucleus, chromatin structure must also be dynamic enough to allow genes to remain accessible and able to mount a rapid transcriptional response when cells are faced with infectious insults or environmental or developmental transitions. Complicating matters is the fact that a large percentage (up to 98%) of human DNA does not encode proteins, and consists of repetitive elements, some of which contain promoters from which transcription can initiate (Forsdyke et al., Trends Immunol., 2002, 23, 575-79). A cell must be able to distinguish desirable versus undesirable gene expression and “self” versus “nonself” nucleic acid material. One structural feature indicating unwanted, nonself, potentially predatory nucleic acid material is presence of double-stranded RNA (dsRNA). Not a usual byproduct of normal gene expression, dsRNA is a component of the life cycle of most viruses. By flagging dsRNA as a sign of unwanted RNA replication, and by avoiding production of dsRNA during most normal gene expression, the cell maintains some level of protection from infection.
RNA interference (RNAi) is an evolutionarily conserved type of epigenetic process in which small dsRNA molecules fully- or partially-complementary to a target nucleic acid induce highly specific gene silencing by triggering the degradation or translational suppression of homologous mRNA. RNAi is believed to represent a form of eukaryotic genome defense from invasion by exogenous sources of genetic material such as RNA viruses and retrotransposons (Carmell et al., Nature Struct. Mol. Bio., 2004, 11, 214-8; Eddy, Nat. Rev. Genet., 2001, 2, 919-929; and Silva et al., Trends Mol. Med., 2002, 8, 505-508).
Sources of triggers for RNAi include exogenously introduced dsRNA, RNA viruses, transposons and endogenous short dsRNAs. In the current model, these triggers are processed by the RNase III enzyme Dicer into small 21-24 nucleotide (nt) short interfering RNAs (siRNAs) which then serve as sequence specific guides to an effector complex called the RNA-induced silencing complex (RISC) that carries out the destruction of homologous mRNAs. Over the past two years, a populous class of endogenous substrates that enter the silencing pathways, the microRNAs or miRNAs, has been described. miRNAs are transcribed from the genomes of diverse organisms, and are reported to lead to suppression of translation, or to target mRNA destruction. In humans, some clustered miRNA genes are transcribed polycistronically as primary precursors (known as pri-miRNAs) that are several hundred bases long. Nonclustered miRNA genes are also predominantly expressed as long nascent transcripts that require further processing, and both the poly- and monocistronic pri-miRNAs undergo a processing step in the nucleus, by another RNase III enzyme, Drosha, that produces shorter, approximately 70-nt pre-miRNAs. The pre-miRNAs are then believed to be exported from the nucleus by exportin-5, a nuclear export factor. Once in the cytoplasm, Dicer processes the pre-miRNAs into mature, approximately 22-nt miRNAs (Carmell et al., Nature Struct. Mol. Bio., 2004, 11, 214-8).
There is evidence that endogenous substrates that enter the RNAi pathway such as the precursors of small non-coding RNAs may undergo RNA editing. RNA editing enzymes have been reported to interact with components of the RNAi pathway. Adenosine deaminases that act on RNA (ADARs) are a class of RNA editing enzymes that deaminate adenosines to create inosines in dsRNA. Inosine is read as guanosine during translation, and thus, one function of editing is to generate multiple protein isoforms from the same gene. ADARs bind to dsRNA without sequence specificity, and due to the ability of ADARs to create sequence and structural changes in dsRNA, ADARs could potentially antagonize RNAi by several mechanisms, such as preventing dsRNA from being recognized and cleaved by Dicer, or preventing siRNAs from base-pairing. Recently, it was shown that the editing of dsRNA by ADARs can prevent somatic transgenes from inducing gene silencing via the RNAi pathway (Knight et al., Mol. Cell, 2002, 10, 809-817). Furthermore, it was recently reported that human and mouse miRNA22 precursor molecules are subject to posttranscriptional modification by A-to-I RNA editing in vivo (Luciano et al., RNA, 2004, 10, 1174-7).
A surge of interest in the RNAi field has resulted in the identification of hundreds of small non-coding RNAs believed to act in gene silencing. One example is a class of small untranslated RNAs, the repeat-associated small interfering RNAs (rasiRNAs), that are associated with repeated sequences, transposable elements, satellite and microsatellite DNA, and Suppressor of Stellate (SOS) repeats, suggesting that small RNAs participate in defining chromatin structure (Aravin et al., Dev. Cell, 2003, 5, 337-350).
Although initially thought of as a purely post-transcriptional process, RNAi appears to act at the transcriptional level as well (Ekwall, Mol. Cell, 2004, 13, 304-5). RNAi may be involved in the formation of heterochromatin at centromere regions which can suppress recombination between homologous repeated DNA sequences. Furthermore, RNAi now believed to play a role in centromere function. A large percentage of small RNAs cloned from the fission yeast, S. pombe, were found to have perfect homology to portions of the centromere region in this organism (Reinhart et al., Science, 2002, 297, 1831), and it was shown that genes involved in the RNA interference pathway are required for pericentromere heterochromatin formation and chromosome segregation (Dawe, Plant Cell, 2003, 15, 297-301; and Volpe et al., Science, 2002, 297, 1833-7). An RNAi effector complex known as the RNA-induced Initiation of Transcriptional gene Silencing (RITS) complex was recently purified and found to be required for heterochromatin assembly in fission yeast. The RITS complex directly links small RNAs produced by Dicer to heterochromatin, because it contains both a previously known chromodomain protein, Chp1, which binds centromeres, and the S. pombe Argonaute homolog (Ago1), which plays a role in RNAi. The RITS complex also contains a previously uncharacterized protein, Tas3, as well as small RNAs that require Dicer for their production. These small RNAs are homologous to centromeric repeats and are required for the localization of the RITS complex to heterochromatic domains, as well as for the methylation of H3 K9 and Swi6 binding to centromeric chromatin, suggesting that RNAi-related processes and small RNAs are involved in epigenetic gene silencing at specific chromosomal loci (Verdel et al., Science, 2004, 203, 6726; and Ekwall, Mol. Cell, 2004, 13, 304-5).
Similar links between RNAi, chromatin modifications, and transcriptional regulation have been established in plants, flies, and the unicellular ciliate, Tetrahymena. In a screen for mutants that suppress silencing in Arabidopsis, the ago4-1 mutant (in the Argonaute family of genes involved in RNAi) was cloned and found to reactivate silenced alleles and decrease CpNpG and asymmetric DNA methylation as well as histone H3 K9 methylation. In addition, the ago4-1 mutant blocked the accumulation of 25-nt siRNAs corresponding to the retroelement AtSN1 (Zilberman et al., Science, 2003, 299, 716-9). In Drosophila, dosage compensation involves epigenetic processes such as the specific acetylation of histones by a histone acetyltransferase, MOF, which specifically binds through its chromodomain to the non-coding RNA roX, an RNA predicted to contribute to chromatin assembly of the dosage compensation complex (DCC) (Akhtar et al., Nature, 2000, 407, 405-9). Also in Drosophila, HP1 localization to heterochromatic regions is dependent on the RNAi machinery (Pal Bhadra et al., Science, 2004, 303, 669-672). In Tetrahymena, RNAi was shown to direct chromatin modifications and DNA elimination (Ekwall, Mol. Cell, 2004, 13, 304-5).
Another role of RNAi in fission yeast is to direct formation of localized repressive chromatin to genes in euchromatin, as was shown by expressing an inverted repeat RNA, which can form a short hairpin RNA (shRNA) capable of targeting heterochromatin formation and cohesin binding in trans. Endogenous, developmentally regulated, lineage-restricted genes (meiotic genes), were also found to be repressed by a similar process involving nearby retrotransposon long terminal repeats (LTRs), implicating interspersed LTRs in regulation of gene expression during cellular differentiation. It was concluded that dsRNA transcripts are acted on by the RNAi pathway to generate siRNAs which trigger the nucleation of a patch of dimethylated H3 K9, Swi6-bound silent chromatin, which can spread outward to silence adjacent genes and attract the evolutionarily conserved Cohesin protein complex mediating sister chromatid cohesion (Schramke et al., Science, 2003, 301, 1069-74). It was further noted that proteins related to CENP-B and the mariner class of transposases also bind pericentric regions of fission yeast centromeres and contribute to the formation of heterochromatin (Shramke et al., Science, 2003, 301, 1069-74; and Nakagawa et al., Genes Dev., 2002, 16, 1766-78).
Some heterochromatin proteins found associated with centromeres and/or telomeres have been shown to be involved in RNA modification and/or RNA degradative processes. Yeast Cbf5p was originally isolated as a low-affinity centromeric DNA binding protein; Cbf5p also binds microtubules in vitro and interacts genetically with two known centromere-related protein genes (NDC10/CBF2 and MCK1). However, Cbf5p was found to be nucleolar and was later found to be involved in transcription and processing of ribosomal RNAs (Jiang et al., Mol. Cell. Biol., 1993, 13, 4884-93; and Cadwell et al., Mol. Cell. Biol., 1997, 17, 6175-83). Cbf5p is a component of a ribonucleoprotein complex that includes box H/ACA small nucleolar RNAs (snoRNAs) and this complex was found to direct the site-specific pseudouridylation of ribosomal RNAs (rRNAs) (Zebarjadian et al., Mol. Cell. Biol., 1999, 19, 7461-72). The RNA component, hTR, of human telomerase, a ribonucleoprotein (RNP) particle required for the replication of telomeres, contains a domain structurally and functionally related to box H/ACA snoRNAs, and hTR is believed to be associated with Dyskerin (the human counterpart of yeast Cbf5p) (Des et al., Nucleic Acids Res., 2001, 29, 598-603). Furthermore, mutations in the human DKC1 gene encoding dyskerin cause dyskeratosis congenita, a rare and fatal inherited syndrome characterized by abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia, and the predisposition to bone marrow failure and malignancies (Mochizuki et al., Proc. Natl. Acad. Sci. U.S., 2004, 101, 10756-61).
Another budding yeast gene, CEP1/CBF1/CP1, is believed to be involved in assembling higher order chromatin structures at centromeres and is associated with a multisubunit complex that processes and degrades RNAs. The CEP1 gene was found to genetically interact with the CSL4/SKI4 gene (Baker et al., Genetics, 1998, 149, 73-85), and the Csl4 protein was later found to be a core component of the exosome, a complex containing multiple 3′-to-5′ riboexonucleases and RNA binding proteins, which is located in both the nucleus and the cytoplasm (van Hoof et al., Mol. Cell. Biol., 2000, 29, 8230-43). In the cytoplasm, the exosome participates in degradation of mRNAs containing AREs (AU-rich elements) such as mRNAs encoding growth factors, cytokines, and proto-oncogene products, whose abundance must be able to change rapidly. Also in the cytoplasm, the exosome has been shown to control the levels of the double-stranded LA virus in yeast. In the nucleus, the exosome is required for trimming the 5.8S rRNA from a 3′-extended precursor, it plays a role in degrading inefficiently spliced or unadenylated pre-mRNAs, and it participates in 3′-end maturation of small nuclear RNAs (snRNAs) and snoRNAs (van Hoof et al., Cell, 1999, 99, 347-50; van Hoof et al., Current Biol., 2002, 12, R285-7; and Butler, Trends Cell Biol., 2002, 12, 90-6).
At least one exosome component has another link to centromere-related function; the fission yeast dis3 gene was originally identified as a mitotic control protein required for sister chromatid separation. The dis3-54 mutation resulted in defective chromosome segregation (Kinoshita et al., Mol. Cell. Biol., 1991, 11, 5839-47; and Ohkura et al., EMBO J., 1988, 7, 1465-73). The S. pombe dis3 gene was later found to be the ortholog of the exosomal protein RRP44, suggesting involvement of nuclear exoribonucleases in chromosome segregation (Shobuike et al., Nucleic Acids Res., 2001, 29, 1326-33). Finally, the RRP44/dis3 gene may be linked to human cancer; human chromosomal region 13q21-q22, containing the KIAA1008 protein (homologous to the fission yeast mitotic control protein dis3), harbors a putative breast cancer susceptibility gene, and has been implicated as a common site for somatic deletions in a variety of malignant tumors (Rozenblum et al., Hum. Genet., 2002, 110, 111-21).
The human homologue of the yeast exosome was recently shown to be the autoantigenic polymyositis/scleroderma (PM/Scl) complex. In addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target six recently identified components of the PM/Scl complex. In sera from patients with idiopathic inflammatory myopathy, scleroderma, or the PM/Scl overlap syndrome, autoantibodies were found to be directed to the human exosome components hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp14p and hCsl4p (Brouwer et al., Arthritis Res., 2002, 4, 134-138).
Most recently, Dicer has been shown to be essential for heterochromatin formation in vertebrate cells. Loss of Dicer results in cell death with the accumulation of abnormal mitotic cells exhibiting premature sister-chromatid separation. Aberrant accumulation of transcripts from alpha-satellite sequences, which consist of human centromeric DNA repeats, was detected in Dicer-deficient cells. Furthermore, two heterochromatin proteins, the Rad21 cohesin protein and BubR1, a cell cycle checkpoint protein, were found to be mislocalized in cells lacking Dicer. It was concluded that the RNAi machinery is involved in the formation of heterochromatin structure in higher vertebrate cells (Fukagawa et al., Nature Cell Biol., 2004, 6, 784-91).